Since the development of high resolution two-dimensional (2-D) electrophoresis by O""Farrell, the technique has been applied to mapping the protein composition of human serum and of various tissues. 2-D electrophoresis consists of isoelectric focusing electrophoresis (IEF) in the first dimension and SDS polyacrylamide gel electrophoresis [SDS-PAGE] in the second dimension. Current interest in using 2-D electrophoresis to identify disease related proteins is exemplified by the existence of databases dedicated to 2-D polypeptide maps of serum and tissue samples of different disease states.
Although 2-D electrophoresis is considered to be the most powerful separation technique for resolving highly complex protein mixtures, the method has limitations. Most of these limitations are related to sample composition, such as high concentrations of salt and protein. The advent of immobilized pH gradient (IPG) strips has greatly minimized these limitations. Even when using immobilized pH gradient strips, however, suggested sample loadings of human serum are on the same order of magnitude (1-5 xcexcL) as that used with the xe2x80x9cclassicalxe2x80x9d O""Farrell technique for analytical 2-D electrophoresis.
The limitation of human serum sample volume is due to the protein distribution and not necessarily the total protein, although total protein is a significant limitation with the O""Farrell technique. A single protein, albumin (HSA), makes up approximately 50% of the total human serum protein. This protein can distort the gel image of a 2-D protein map when large sample volumes are used. The limitation in sample volume ultimately limits the number of proteins that can be detected by 2-D electrophoresis.
The distortion in the gel image is particularly evident in the area of the albumin (molecular weight [MW] 66,000, pI 4.9) where vertical and horizontal streaking masks a large portion of the protein map. In addition, a group of abundant serum proteins, immunoglobulin (Ig), contributes approximately 20% to total human serum protein. Vertical and horizontal streaking also masks the portion of the protein map in the area of the gel image where Ig light and heavy chains are located. Furthermore, the presence of the abundant HSA and Ig alters the pI of the isoelectric focusing electrophoresis gel in these proteins, impeding effective resolution and detection of many other protein spots. To improve 2-D electrophoresis human serum maps, in both quality of image and the number of detectable proteins, human serum albumin must be specifically removed.
There are currently several methods for removing albumin from serum, such as adsorption to activated carbon particles, binding to Cibacron-blue dye coupled to Sepharose beads, and the use of anti-albumin polyclonal antibodies. Removal of serum albumin using carbon or the Cibacron-blue Sepharose is relatively inexpensive, but these methods suffer from a lack of specificity. The Cibacron-blue dye binds many proteins other than albumin, such as interferon, lipoproteins, blood coagulation factors, kinases, dehydrogenases and most enzymes requiring adenyl-containing cofactors. Also, because of the microporous nature of the Sepharose beads, additional proteins are trapped in the dead volume of the rigid matrix.
The present invention provides a method for removing interfering macromolecules from a liquid sample before protein fractionation. This method involves contacting the liquid sample with a polypeptide affinity reagent that has specificity for an abundant macromolecule in the sample, and is one member of a high affinity binding pair system. A macromolecule-polypeptide affinity reagent complex is formed, that is then contacted with the other member of a high affinity binding pair system to form a xe2x80x9ccollapsible affinity matrix.xe2x80x9d The collapsible affinity matrix is specific for the abundant macromolecule and, when centrifuged, contains very little dead volume that would otherwise trap additional sample macromolecules. In one embodiment, the invention provides a method for specifically removing macromolecules from a sample using biotinylated adsorptive proteins. In a specific embodiment, a biotinylated anti-HSA antibody, in conjunction with avidin and human serum, forms a collapsible affinity matrix, containing albumin. The combination of biotinylated protein A, avidin, and human serum, followed by contact with biotinylated anti-HSA and avidin allows simultaneous co-precipitation of albumin and immunoglobulin (Ig). The practice of the method of the invention can thereby provide serum samples substantially depleted of albumin and immunoglobulin.
The invention also provides a monoclonal antibody (HSA2126NX.012) that can specifically immunoprecipitate albumin from serum. The invention further provides a kit useful for specifically removing abundant macromolecules from a sample using biotinylated adsorptive proteins.
This unique method for removing albumin and immunoglobulin from serum permits the full potential of the powerful protein fractionation technique of high resolution 2-D electrophoresis to be attained, by making possible visualization of low abundant serum proteins, as well as those proteins that would normally be obscured by the serum albumin and immunoglobulin. This advantage allows for identification and characterization of a variety of novel markers that may have diagnostic or therapeutic utility. For example, the discovery of novel biochemical serum markers for the diagnosis of various disease states such as osteoporosis, arthritis, cancer or cardiovascular disease can aid immensely in the management of these conditions.
The removal of high abundant macromolecules from a liquid sample followed by a high resolution 2-D electrophoresis allows for visualization of low abundant sample proteins that might not be visualized with limits in total protein load. When the high resolution 2-D electrophoresis includes in-gel sample rehydration of immobilized pH gradient strips, followed by isoelectric focusing in the first dimension and SDS-PAGE in the second dimension, this method is called xe2x80x9cHigh Capacity Two-Dimensional Polyacrylamide Gel Electrophoresisxe2x80x9d (xe2x80x9cHiCap 2-D PAGExe2x80x9d). HiCap 2-D PAGE permits relatively high amounts of low abundant proteins to be loaded following the removal of albumin and immunoglobulin. HiCap 2-D PAGE also permits the use of large sample load due to in-gel sample rehydration. The combination of abundant serum protein removal by the collapsible affinity matrix and HiCap 2-D PAGE produces highly reproducible maps of low abundance serum proteins in human serum.